BackgroundPsoriasis is a genetically determined inflammatory and proliferative disease of the skin. There is now increased evidence for the role of nitric oxide in some diseases, including psoriasis. Nitric oxide is a potent regulator of keratinocyte growth and differentiation and stimulates angiogenesis.AimThis study aimed to detect serum nitric oxide level in active psoriasis patients, to correlate these levels with severity of the disease scored with psoriasis area and severity index, and compare them with those in normal individuals.Patients and methodsTwenty-five patients with active psoriasis and 25 age-matched and sex-matched controls were recruited after their written consent was obtained. Patients on local or systemic treatment or with coexisting inflammatory skin disease were excluded. Serum nitric oxide levels were assessed using the Griess method.ResultsThe mean serum nitric oxide levels (95.96 ΁ 12.7 ΅mol/l) were significantly higher in active psoriasis patients than that in the healthy controls (27.1 ΁ 6.85 ΅mol/l).ConclusionThe significantly elevated serum levels of nitric oxide and their positive correlation with the severity of psoriasis may suggest the possible role of this mediator in the etiopathogenesis of the disease, and suggest a potential future therapy for the disease.

Psoriasis is a genetically determined inflammatory and proliferative disease of the skin. It is a common chronic, recurring skin disease that is characterized by macroscopic and microscopic skin alterations [1] .

The pathogenesis of psoriasis can be considered in the context of three phases: phase I is an interaction between genetic and environmental factors; phase II is an interaction between innate/adaptive immunity and the resident skin cells; and phase III consists of epidermal and dermal remodeling (characterized by the psoriatic plaque) [2] .

At the cellular level, psoriasis is characterized by markedly increased epidermal proliferation and incomplete differentiation, elongation, dilatation, and 'leakiness' of the superficial plexus of dermal capillaries and a mixed inflammatory and immune cell infiltrate of the epidermis and papillary dermis [3] .

There is now little doubt that nitric oxide (NO) is a novel and powerful signaling molecule that induces many types of cellular responses and affects the functions of most organ systems. Whereas the role of NO in many biologic systems is now well documented, we are still just beginning to understand how NO acts within the molecular cascade of cell growth and cell death, inflammation, and immune response in human skin. It is already apparent that this molecule is involved in normal physiologic processes of the skin by serving regulatory functions in vascular homeostasis, melanogenesis, and responses to ultraviolet irradiation. Increasing evidence exists for a critical role of NO in pathologic processes of the skin. These include psoriasis and other immune-mediated skin diseases [4] .

As a potent regulator of keratinocyte growth and differentiation, multifunctional signaling NO has been considered to be a strong candidate in the pathogenesis of psoriasis [5] .

NO may trigger the psoriatic disease process at least partly through an increase in the release and actions of calcitonin gene-related peptide and substance P, which are considered to play important roles in the pathomechanisms of psoriasis by inducing the production of adhesion molecules, keratinocyte hyperproliferation, mast cell degranulation, vasodilatation, and chemotaxis of neutrophils [6] .

In animal tissues, NO is generated enzymatically by synthases [nitric oxide synthase (NOS)], which oxidize l-arginine to l-citrulline [7] . There are three isoforms of NOS: NOS I or nNOS - the neuronal form; NOS II or iNOS - inducible NOS present in various cell types upon inflammatory stimulation (e.g. macrophages); and NOS III or eNOS - the constitutive enzyme primarily discovered in the endothelium [8] . In the skin, NO is produced by iNOS in several cells, namely in keratinocytes, fibroblasts, Langerhans cells, and other dendritic cells [9] .

NO is vital to the activity of proangiogenic cytokines. Vascular endothelial growth factor (VEGF) is the most potent angiogenic factor in psoriasis, and this effect involves the modulation of NO [10] . VEGF increases NO production by upregulation of eNOS and, conversely, the angiogenic effect of VEGF appears to be dependent on NO [11] . Pharmacological blockade of NOS prevents both VEGF-induced endothelial cell proliferation and VEGF-mediated activation of mitogen-activated protein kinases [12] . VEGF-stimulated endothelial cell migration and decreased adhesion, and organization are also dependent on NO. These effects may rely on iNOS as well as eNOS [13] .

Characterization of these intrinsic and extrinsic regulatory stimuli of NO synthesis has yielded considerable insights into the role of NO in inflammation and hyperproliferation that occur in psoriasis, and may ultimately form the basis for future therapeutic intervention. The demonstrable and potential roles of NO in psoriasis pathogenesis are the subjects of this study. This was assessed by detecting serum NO levels in patients with active psoriasis and correlating these levels with severity of disease and comparing them with those in normal individuals.

Patients and methods

This case - control study was carried out on 25 patients with clinically active psoriasis and 25 healthy controls; the two groups were similar in most demographic characteristics and were recruited from the outpatient clinic of the Department of Dermatology and Venereology, Suez Canal University Hospital. The study was approved by the local ethics committee of the Faculty of Medicine, Suez Canal University. Our study included patients with four types of psoriasis (plaque, guttate, erythrodermic, and arthropathic). Informed consent was obtained from all the participants after a full explanation of the study was provided to them.

Detailed assessment of history was performed including duration of disease and duration of current episode. Clinical examinations and investigations including complete blood picture (CBC) liver and renal functions tests were carried out. The assessment of the severity and extent of disease was carried out using the psoriasis area and severity index (PASI) score. Pregnant or lactating women, patients with coexisting inflammatory skin disease, patients who had received topical or systemic therapy within the last 4 weeks, those with systemic illnesses such as Rheumatoid Arthritis (RA), Systemic Lupus Erythematosus (SLE), or malignancies, and patients taking sildenafil citrate regularly were excluded from the study.

Five milliliter of venous blood was collected and placed in plain tubes that were allowed to clot for 30 min and then centrifuged for 5-10 min to obtain serum. The serum samples collected were stored at -20°C until analysis in one run. As NO is an unstable molecule, it is rapidly converted into nitrates and nitrites in the body; hence, their concentration is parallel to NO levels.

Nitrite and nitrate were measured using a Griess method [14] . In this method, nitrate is reduced enzymatically with nitrate reductase into nitrite [15] , which is treated with sulfanilamide and N-1-naphthyl-ethylene diamine. A red-colored compound is formed; its characteristic absorption spectrum was determined using spectrophotometry. Results are presented as μmol/l.

Statistical analysis was carried out using SPSS (SPSS Inc., Chicago, Illinois, USA) computer software tests. The differences in NO levels among patients and controls were tested using the Student t-test to compare the mean values, considered significant when P value was less than 0.05. The comparative analysis among ratios was carried out using the analysis of variance test, with significant difference indicated when P value was less than 0.05.

Results

This study included 50 individuals divided into two groups: 25 patients had psoriasis and the other 25 individuals were age-matched and sex-matched healthy controls. Their demographic data are shown in [Table 1]. The severity of psoriasis of the patients studied was distributed according to the PASI score into mild, moderate, and severe as shown in [Table 2].

Table 1: Distribution of the patients studied according to sex and age

Statistical analysis of the mean NO level in the patients studied versus the mean PASI score indicated a statistically significant increase in the NO level as the PASI score increased as shown in [Table 4].

Patients with four types of psoriasis were included in this study: the mean NO levels were higher in erythrodermic and arthropathic patients, but the numbers of both erythrodermic and arthropathic patients were so low that it was difficult to draw any significant conclusion as shown in [Table 5].

Table 5: Mean nitric oxide levels in patients with different types of psoriasis

There was a statistically significant positive correlation between NO levels and severity of psoriasis measured by the PASI score in the patients studied (Pearson's correlation r = 0.9) as shown in [Figure 1].

There is no doubt that advances in the understanding of the cellular immunology and biology of psoriasis, when coupled with the revolution in biotechnology and rapid advances from human genetic studies of autoimmunity, have enhanced insights into the cause and treatment of psoriasis [16] .

In recent years, the clonality of lymphocytes in psoriatic lesions and the increased cytokine release in that region have been reported extensively. Cytokines, such as IFNγ, TNFα, IL-8, IL-1, and IL-6, are the most important ones. Recently NO is considered to be one of these mediators of inflammation [17] .

The aim of this study was to estimate the levels of NO in different types of psoriasis, compare them with normal individuals, and correlate them with the severity of disease. In the study of serum NO levels in psoriasis carried out by Gokhale et al.[18] , it was found that NO levels were significantly increased in patients with psoriasis and these levels showed a positive correlation with the severity and duration of the disease.

Zalewska et al. [19] also found higher plasma NO levels in psoriatic patients in comparison with controls. They reported a positive correlation between PASI and NO levels and thus considered NO to be an important element in the inflammatory process in psoriasis and in the maintenance of lesional erythema.

Similarly, Orem et al.[20] studied NO levels in patients with psoriasis during active and inactive phases. They observed increased NO production in patients with a mean PASI of 16.5 and above. They also found a positive correlation between NO levels and the activity of disease. During the inactive phase (mean PASI 1.7), the NO levels were low. Another supporting study was carried out by Kadam et al.[21] in which NO was significantly higher in patients with active psoriasis compared with healthy controls; moreover, NO levels were significantly correlated with the severity of the disease.

In our study, we found significantly high serum NO levels in patients with active disease compared with normal individuals. However, although the mean levels of NO increased as the duration of the disease increased, this was statistically nonsignificant.

Evidence suggests that NO promotes vasodilatation and vascular permeability, activates cyclo-oxygenase, and stimulates the production of TNFα. Concentrations of NO have cytotoxic and pathophysiological functions; low concentrations, as produced by vascular endothelium, are believed to play a protective role in the microvasculature [22] .

Other supporting results were derived from measurement of NO locally in the skin such as the study carried out by Kolb-Bachofen et al.[23] , who found increased expression of iNOS in psoriatic plaque, concluding that iNOS expression is involved in the pathogenesis of cutaneous inflammation of psoriasis.

Ormerod et al.[24] evaluated punch biopsy specimens from lesional and nonlesional skin regions of patients with psoriasis and normal controls. They studied the presence of endothelial, neuronal, and inducible isoforms of NOS using immunocytochemistry with computerized image analysis. In addition, direct NO production on plaques with psoriasis and nonlesional areas was assessed in a separate group of psoriatic patients. Significantly increased neuronal isoform NOS levels were found in psoriatic epidermis compared with normal skin. Inducible isoform NOS levels were also significantly increased in lesional compared with clinically uninvolved skin. NO production from the skin surface (direct measurement) was 10 times higher in the lesions of patients with psoriasis compared with those with normal skin.

Cals-Gierson et al. [25] and Ormerod [26] have reported that NO is also known to stimulate epithelial cells to produce and release chemokines and other growth mediators such as VEGF, which appear to be important for keratinocyte proliferation and angiogenesis. They have also reported that the increased NOS2 expression observed in various inflammatory conditions such as dermatitis may be responsible for the impaired barrier function found in these situations. The events leading to this are the elevated levels of NO and peroxynitrite formation, which in turn cause increased activation of poly-(ADP ribose) polymerase, which further translates into inhibition of keratinocyte differentiation. This could be one of the postulated mechanisms of psoriasis pathogenesis. NO is also found to increase the level of cGMP, which may act as a secondary mediator and induce proliferation of keratinocytes [25] . Ormerod et al.[26] showed that application of an NO-releasing cream to normal skin produced an increase in markers for T-lymphocytes and endothelial cells, both of which are features of psoriasis. They also found decreased NO production in psoriatic plaque after the application of iNOS inhibitor - NG monomethyl-l-arginine. However, Morhenn [27] reported deterioration in psoriatic plaque after the application of an NO donor, that is, nitroglycerine.

iNOS is found in inflammatory conditions and has recently been observed in psoriasis. It has been shown that the quantity of NO released from psoriatic skin is between 100 and 1000 times higher than that released from normal skin. Several events and cell types implicated in the pathogenesis of psoriasis, including T-lymphocyte function, cytokine release and activity, and cell proliferation and differentiation, are regulated at least partly by NO [9] .

However, Nilgun et al.[28] found a significant increase in serum NO levels in psoriatic patients, but found no correlation between NO and PASI score. Also, Abeyakirthi et al.[29] found that NO is decreased in psoriatic skin; this could be attributed to the fact that low concentrations of NO induce keratinocytes proliferation, whereas high concentrations induce differentiation, explaining the fact that psoriatic keratinocytes are poorly differentiated and hyperproliferative.

We suggest that more studies need to be carried out on the effects of different therapeutic modalities of psoriasis on serum and local tissue levels of NO as characterization of the role of NO in cutaneous disease will not only contribute toward our understanding of cutaneous biology but is also likely to be the foundation for the development of novel therapeutic approaches that can modify, arrest, or reverse the course of the disease.

In conclusion, this study shows that serum NO level is significantly higher in patients with psoriasis than in normal individuals, which supports the role of NO in the pathogenesis of psoriasis.